PROJECT SUMMARY/ABSTRACT Chagas disease (ChD) is a parasitic disease caused by the infection with the protozoan Trypanosoma cruzi. It is estimated that 6million people are infected by T. cruzi worldwide, with 70million at risk of infection. The majority of T. cruzi-chronically infected individuals remain in an asymptomatic clinical form, named indeterminate, while about 30% of the patients develop a severe cardiomyopathy that leads to over 10,000 deaths/year. There is no vaccine to prevent ChD, nor interventions that can prevent the progression of cardiomyopathy. ChD cardiomyopathy is the consequence of an inflammatory reaction, which leads to tissue destruction and pathology. Despite the complexity of the host-parasite interaction that leads to cardiomiopathy, it is a consensus that host's immune response is critical in determining disease outcome. We have previously shown that the indeterminate and cardiac clinical forms of ChD are associated with a predominant expression of down modulatory and inflammatory cytokines, respectively. We have also shown that CD4-CD8- (double- negative ? DN) T cells are major sources of these cytokines in patients with chronic ChD (CChD). In addition, we have demonstrated that in vitro blocking of T. cruzi-induced DN T cell activation, through the inhibition of antigen presentation via CD1d, shifts the cytokine expression of DN T cells from an inflammatory to a predominantly anti-inflammatory profile. Thus, we identified, for the first time, a cell population that may be a potential target for an immunotherapeutic approach to inhibit inflammation-induced pathology and prevent cardiomyopathy development. The main objective of this project is to elucidate cellular and molecular mechanisms behind the activation of DN T cells during CChD, with the long-term goal of designing strategies to prevent development of cardiac pathology. Our specific aims are: (1) To identify the T. cruzi-derived antigen(s) responsible for the activation of DN T cells and identify the DN T cell subpopulation most reactive (TCR ?/?, TCR ?/?, and NK T cells) to this(ese) antigen(s) from indeterminate and cardiac Chagas patients with different degrees of cardiomyopathy; (2) To identify the antigen-presenting molecule responsible for presentation to DN T cells and to test if blocking of the antigen-presenting complex will modulate the activation and functional profile of DN T cell subsets from Chagas patients of different clinical forms and stages; (3) To analyze the coding transcriptome of purified DN T cell subsets from distinct Chagas patient groups before and after blocking the activation by the specific parasite component. By bringing together a group of Brazil and US- based scientists with complementary expertise, we hope to dissect the role of DN T cells in disease progression, working towards the development of immunological interventions, finding biomarkers of disease progression, while performing technology transfer and scientific training amongst all involved institutions.